Proteomic analyses were performed to identify regulated liver proteins in rainbow trout (Oncorhynchus mykiss) caged upstream and downstream from a sewage treatment works (STW). Two-dimensional gel electrophoresis, image analysis and FT-ICR mass-spectrometry revealed four regulated protein spots. The three down-regulated spots contained betaine aldehyde dehydrogenase, lactate dehydrogenase and an unidentified protein respectively. The only up-regulated spot consisted of both mitochondrial ATP synthase alpha-subunit and carbonyl reductase/20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD). Further studies using quantitative PCR revealed a 13.5-fold induction of CR/20beta-HSD B mRNA following STW effluent exposure. The CR/20beta-HSD B gene was not regulated by 17alpha-ethinylestradiol, suggesting that its induction downstream from the STW is due to other factors than exposure to estrogens. Image analysis was initially performed on four gels from each group. These analyses suggested 15 regulated spots. However, validation of the 15 spots by increasing the number of replicates confirmed only four regulated spots. Hence, the present study also demonstrates the need for sufficient biological/technical replication in the interpretation of proteomic data.

Progesterone is a survival factor in rat periovulatory granulosa cells. The mechanisms involved are unclear but progesterone receptor (PGR) antagonists have been shown to inhibit cholesterol synthesis and induce apoptosis. Furthermore, reports suggest that statins induce apoptosis by inhibition of protein isoprenylation. Statins inhibit the rate-limiting step of the cholesterol synthesis, thereby reducing availability of intermediates used for the post-translational isoprenylation process. It has been suggested that PGR antagonists in a similar manner induce apoptosis by decreasing cholesterol synthesis and thereby protein isoprenylation. In this study we hypothesized that the mechanism by which the nuclear PGR antagonist Org 31,710 induces apoptosis in rat periovulatory granulosa cells, is by decreasing cholesterol synthesis and thereby general cell protein isoprenylation. Incubation of isolated granulosa cells with Org 31,710 or simvastatin for 22 hr resulted in increased apoptosis and reduced cholesterol synthesis. However, simvastatin caused a substantial inhibition of cholesterol synthesis after 6 hr in culture without inducing apoptosis. In contrast, Org 31,710 had only a modest effect on cholesterol synthesis after 6 hr while it significantly induced apoptosis. Addition of isoprenylation substrates partially reversed apoptosis induced by simvastatin and to a lesser extent apoptosis induced by Org 31,710. In addition, and in contrast to Org 31,710, simvastatin caused a decrease in isoprenylation of a selected isoprenylation marker protein, the Ras-related protein RAB11. In conclusion, we demonstrate that the PGR antagonist inhibits cholesterol synthesis in granulosa cells but reduced protein isoprenylation is not the mediating mechanism of increased apoptosis as previously hypothesized.